Separation of platelets from whole blood for use as a healant

ABSTRACT

A method is described for separating, retrieving and concentrating platelets from whole blood relying on aggregation of the platelets followed by filtration. This method eliminates the need of a centrifuge for separating said cells from blood. To obtain cellular concentrates of platelets, blood is mixed with compatible agents that will aggregate cells while retaining contained growth factors. The resulting aggregates can then be separated from blood by filtration. If desired, the filter-captured aggregates are subject to a brief washing cycle where they are washed clean of residual aggregating agent, plasma, and red cells. Aggregates can then be partly or wholly deaggregated and the cells retrieved. The result is a suspension of cells and small aggregates with therapeutic levels of concentrated blood cells with included growth factors that are available for delivery to a wound site. A device that accomplishes the aforementioned process is also described.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/289,224 filed on May 7, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates to wound healants, as well assystems, methods, and devices for harvesting growth factors. Morespecifically, by separating platelets from whole blood, growth factorrich platelets can be harvested, concentrated, and delivered for woundhealing in accordance with embodiments of the present invention.

BACKGROUD OF THE INVENTION

[0003] An attractive emerging clinical approach for augmenting woundhealing is the rapidly expanding clinical and surgical use ofrecombinant or autologous growth factors for improved therapeuticoutcomes. Examples of areas where such wound healing compositions areuseful include intractable decubitus and pressure ulcers; orthopedicbone defect repair and bone ingrowth in fixation and implantationprocedures; plastic and maxillofacial surgery; burn skin grafts;connective tissue repair; periodontal surgery, etc., as described by:Knighton D R, Surgery, Gynecology & Obstetrics 170: 56-60. 1990; and inSlater M, J Orthop Res 13: 655-663. 1995. The widespread clinical andsurgical acceptance of growth factor-based wound healing therapies arecurrently limited to some degree by the high cost associated with bothrecombinant and autologous growth factor healants, and the additionalinconvenience of processing autologous cells intraoperatively. Althoughonly few controlled comparisons have been made between autologous growthfactor cocktails and purified protein recombinant growth factors forwound healing effectiveness, a single recombinant growth factor may beless effective in many wound healing applications than a combination ofnatural growth factors (PDGF, VEGF, TGF, EGF, etc.) present in plateletsas suggested by Cromack D T, J Trauma. 30: S129-S133, 1990. To that end,several potentially therapeutic growth factor compositions have beendeveloped that contain more than one growth factor. However, theclinical applicability of some of these healants can be limited by highcost and inconvenience of obtaining growth factor compositions for use.

[0004] The process described herein would allow for a cost effective,timely, and convenient technique for isolation and concentration ofautologous or homologous platelets with included active growth factors.These cells may be harvested from small volumes of patient bloodpreoperatively, intraoperatively, perioperatively, or for outpatientprocedures to allow for convenient and sustained delivery of growthfactors to effectively promote healing. Current autologous growth factorharvesting systems are expensive and rely on technician-intensive bloodprocessing to generate platelet rich plasma (PRP) prior to theadditional steps required to harvest platelets to desired therapeuticconcentrations, e.g., by centrifugation, by gel filtration, or byultrafiltration. Laboratory separation by gravity has also been used,though time constraints makes this a less desirable approach.

SUMMARY OF THE INVENTION

[0005] It has been recognized that providing a wound healant usingsystems, methods, and devices to rapidly, conveniently andcost-effectively harvest platelets from whole blood using aggregation ofplatelets followed by filtration of those aggregates and appropriateretrieval would be an advancement in the area of accelerated woundhealing. Such autologous platelet harvesting methods and systems canenable the elimination of the use of a centrifuge that can be expensive,inconvenient to operate, and can be more time consuming to use than theuse of the systems, methods, and devices of the present invention.

[0006] With this in mind, a method for separating platelets from wholeblood can comprise the steps of (a) providing whole blood includingplatelets, leukocytes, erythrocytes, and blood plasma; (b) selectivelyaggregating platelets using an aggregating agent to form plateletaggregates that are larger in size than said leukocytes anderythrocytes; and (c) substantially separating the platelet aggregatesfrom the leukocytes, erythrocytes, and blood plasma. Alternatively, amethod for separating platelets from a platelet suspension can comprisethe steps of (a) selectively aggregating platelets using an aggregatingagent to form platelet aggregates in a platelet suspension where theaggregation function of platelets is realizable; and (b) separating theplatelet aggregates from platelet suspension by filtration. The plateletsuspension can be whole blood, platelet rich plasma, or a platelet pack,for example.

[0007] In either of the above embodiments, the platelet aggregatesobtained can then be washed, deaggregated, and/or suspended afterseparating, such as by filtration, in a desired solution. In oneembodiment, the platelet aggregates can be deaggregated and concentratedto a desired therapeutic level for delivery to a wound site. Further,though centrifugation is not precluded by the present method, in apreferred embodiment, the platelets can be separated without the use ofcentrifugation. Additionally, the step of selectively aggregating theplatelets can be enhanced by mixing the whole blood and the aggregatingagent at a temperature from 20° C. to 37° C. for about 60 to 180seconds.

[0008] Many aggregating agents can be used in accordance with thepresent invention, including aggregating agents selected from the groupconsisting of thrombin, ristocetin, arachidonic acid, collagen,epinephrine, adenosine diphosphate, and combinations thereof. Apreferred aggregating agent for use is adenosine di-phosphate (ADP).

[0009] In a more detailed aspect of the present invention, the plateletscan be washed with a desired salt solution at a temperature from about18° C. to 30° C. and for about 1 to 3 minutes to remove residualcomponents including agonists, red blood cells, and plasma proteins. Inanother more detailed aspect, an additional step can be carried out thatincludes gently agitating/aspirating the aggregated platelets afterseparation with an appropriate solution to deaggregate and resuspend theplatelets while minimizing degranulation, thereby recovering singlecells and/or small cell aggregates. With this embodiment, theagitation/aspiration step can occur under controlled temperatures from33° C. to 37° C., and at a pH from about 6 to 8.

[0010] Once the platelets are collected, they can be applied to a wound,target tissue, or cells by any of a number of methods. For example, thestep of filtering can be conducted with a biodegradable filter that canbe placed directly on a wound site. Such a biodegradable filter cancomprise a material selected from the group consisting of polyglycolicacid, polylactic acid, polypeptides, collagen, hyaluronic acid andcombinations thereof. Alternatively, suspended cells can be placeddirectly on a wound site, such as by a direct application, apoly-glycolic acid patch, a polyester patch, or a gel seal suture.Furthermore, platelets can be mixed with plasma, gelatin,fibrin/fibrinogen, alginates, chitosan or other sponges, bone fillerssuch as calcium phosphate, or other similar clinically accepted wounddressing/filling substrates.

[0011] One additional aspect of the present invention that is desirableis the fact that autogolous treatment can occur within 15 minutes orless following blood collection. This is particularly useful foremergency reinfusion or transfusion of platelets. This is also useful inoperating room settings where time is often critical during surgicalprocedures in which a wound healant is desired for use.

[0012] In another embodiment, a device for separating platelets fromwhole blood can comprise a mixing/filtering chamber configured formixing whole blood and an aggregating agent when positioned in a firstorientation, and further configured for filtering platelet aggregatesformed during mixing when positioned in a second orientation. The devicecan also contain an inlet or multiple inlets for transferring wholeblood and the aggregating agent into the mixing/filtering chamber(together or separately), and washing the aggregates. The device canalso comprise a mixing mechanism for mixing the whole blood and theaggregating agent when the mixing/filtering chamber is positioned in thefirst orientation, and a filter for collecting the platelet aggregateswhen the mixing/filtering chamber is positioned in the secondorientation. One or more outlet ports can also be present for removingresidual components of the whole blood that are not collected on thefilter, and to provide a means to access, deaggregate and harvest theaggregates captured on the filter. In one embodiment, the mixingmechanism can be configured to optimize mixing, and to preventsubstantial premature release of growth factor contents from theplatelets, e.g., by means of a magnetic stirrer.

[0013] In an alternative device embodiment, a device for separatingplatelets from whole blood can comprise a mixing chamber configured forreceiving and mixing whole blood and an aggregating agent to formplatelet aggregates and residual blood components. A filtering chambercan be configured for collecting platelet aggregated in the mixingchamber and allowing the residual blood components to substantiallypass, wherein the filtering chamber comprises a porous filter. A valvecan be placed between the mixing chamber and the filtering chamber forholding the whole blood and the aggregating agent in the mixing chamberduring mixing, and for allowing flow of the platelet aggregates and theresidual blood components from the mixing chamber in to the filteringchamber to separate aggregates from blood. Additionally, an outlet orfilter port can be present and configured for removing components of thewhole blood that are not collected in the filter.

[0014] The filter used in either of the above device embodiments can bemade of single or multiple stages of filters with a pore size rangingfrom 15 to 500 μm, though a more preferred pore size can range from 15to 100 μm. In one embodiment, the filter can comprise a materialselected from the group consisting of metals, polymers, biomaterials,biodegradable materials, and combinations thereof. In more detail, thefilter can comprise a material selected from the group consisting ofstainless steel, nylon, poly-tetra-fluoro-ethylene (Teflon), polyester,and/or hyaluronic acid. The outlet port can also act as an inlet portfor injecting a physiological medium for washing and/or for injecting adeaggregating agent, though this is not required. In further detail withrespect to preferred structures, the mixing device can comprise anelectromagnetic motor and a magnetic stir bar.

[0015] In still another embodiment, a wound healant for human tissue cancomprise a physiological solution, essentially free of thrombin or otherdegranulating agents, autologous platelets suspended in thephysiological solution; and a clinical carrier substrate configured forcarrying the platelets to a wound site. Such a solution is merely onepossible composition that can be prepared in accordance with the methodsof the present invention. However, this composition can be desirable foruse because wound treatment can be effected without the step of adding adegranulating agent, e.g., thrombin, that are typically used in the art.

[0016] Alternatively, a wound healant for application to tissue cancomprise a combination of platelet aggregates and single platelets,wherein the platelet aggregates and the single platelets are suspendedin a physiological solution and carried by a clinical carrier substrate.An advantage of utilizing such a combination includes the possibilitythat aggregated platelets may provide immediate release of growth factorto a tissue, whereas individual, non-aggregated platelets may providegrowth factor to a tissue site over time. Therefore, such a woundhealant can provide immediate growth factor treatment to a tissue, aswell as provide some sustained release of growth factor over time.

[0017] In either of the above embodiments, substantially intactplatelets can be delivered directly in, for example, a physiologicalsolution such as isotonic saline solutions. In one embodiment of such awound healant, the autologous platelets with contained growth factorscan be present at a concentration greater than three times normal levelscompared to that present in blood, wherein the platelet is notsubstantially activated by degranulating agents such as thrombin.Further, the physiological solution that acts as the wound healant canbe essentially free of serum or plasma proteins.

[0018] As described herein with respect to a process of the presentinvention, the platelets can be prepared and be present as single cellsor small aggregates of cells that are distributed within a clinicalcarrier substrate, thereby providing a dispersed and prolonged releaseof growth factors. The healant can be delivered as suspended cells in aphysiological solution, in a patch such as a poly-glycolic acid patch, apolyester patch such as a Darcon patch, or as a gel such as part of agel suture system. Additionally, such a wound healant can also becombined with a hemostatic sealant to contribute growth-promotingproperties of the wound healant. Further, such a wound healant can alsobe incorporated into aneurysm substrates or fillers such as coils orgels to accelerate the healing and/or re-integration of an aneurysm.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In the accompanying drawings which illustrate embodiments of theinvention:

[0020]FIG. 1 is a perspective view of a system in accordance with anembodiment of the present invention;

[0021]FIG. 2 is front view of a mixing/filtering chamber of the systemof FIG. 1, and is configured in the mixing position;

[0022]FIG. 3 is front view of a mixing/filtering chamber of the systemof FIG. 1, and is configured in the separating position;

[0023]FIG. 4 is a schematic representation of an alternative embodimentof the present invention wherein the mixing and filtering chamber areseparated by and valve;

[0024]FIG. 5 is a graphical representation showing a hemocytometer countof platelet yield of a recovered cell suspension compared to whole bloodand filtrate;

[0025]FIG. 6 is a graphical representation comparing function ofplatelets recovered by the aggregation/filtration process of the presentinvention and function of platelets recovered using a conventionalcentrifugation technique of the prior art; and

[0026]FIG. 7 is a graphical representation comparing PDGF-AB recoveryfrom the aggregation/filtration process of the present invention andPDGF-AB recovery using a conventional centrifugation technique of theprior art.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

[0027] Reference will now be made to the exemplary embodimentsillustrated in the drawings, and specific language will be used hereinto describe the same. It will nevertheless be understood that nolimitation of the scope of the invention is thereby intended.Alterations and further modifications of the inventive featuresillustrated herein, and additional applications of the principles of theinventions as illustrated herein, which would occur to one skilled inthe relevant art and having possession of this disclosure, are to beconsidered within the scope of the invention. It is also to beunderstood that this invention is not limited to the particularconfigurations, process steps and materials disclosed herein as thesemay vary to some degree. It is also to be understood that theterminology used herein is used for the purpose of describing particularembodiments only, and is not intended to be limiting as the scope of thepresent invention.

[0028] It must be noted that, as used in this specification and theappended claims, singular forms of “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise.

[0029] “Physiological solution” or “physiological medium” can be saline,plasma, or other isotonic salt solutions.

[0030] As illustrated in FIG. 1, a system, indicated generally at 10, inaccordance with an embodiment of the present invention is shown. Inaccordance with one aspect of the present invention, the system 10provides a base 12 and a well 14 within the base. Extending upwardlyfrom the base 12 is a longitudinal support 16 terminating in a flatupper surface 15 containing an aperture 17 defined by support 16 and apair of partially encircling arms 18. Aperture 17 is configured forholding cylindrical mixing/filtering chamber 20.

[0031] The cylindrical mixing/filtering chamber 20 has an enclosedexpanded end consisting of a flange 22. The flange 22 is larger than thediameter of aperture 17 such that when chamber 20 is inserted intoaperture 17 in an inverted filtering or separating position, as shown,the flange 22 will rest on flat surface 15. A space 19 between arms 18is provided to enable a syringe 44 (not shown) or other device attachedat the opposite end of mixing chamber 20 to pass between space 19 whenthe mixing chamber is inserted into or removed from aperture 17. Thewell 14 is also configured to hold the mixing chamber 20 at the flangedend 22. Specifically, the flanged end 22 of the mixing/filtering chamber20 can be placed in the well 14 in a mixing position (not shown). Inthis position, the mixing/filtering chamber 20 is in position for gentlymixing the whole blood. Thus, the system 10 provides a means of fixingthe mixing/filtering chamber 20 in both a filtering position as shown,and in a mixing position (not shown).

[0032] The mixing/filtering chamber 20 is described in greater detailhereinafter. FIG. 1 shows a filter 24 for filtering aggregated bloodcells, a stem 28 for removing and adding fluids, and a valve 30 forstarting and stopping fluid flow. With respect to the mixing/filteringchamber 20, the outside surface of the cylindrical walls can containtongues and/or grooves (not shown) and arms 18 can likewise containmatching grooves and/or tongues (not shown) such that, when the mixingdevice is inserted in aperture 17, it will be locked in a tongue ingroove non-rotating position. Likewise, a similar system can be presentwhere the mixing/filtering chamber 20 rests in the well 14.

[0033] In FIG. 2, the mixing/filtering chamber 20 is shown in a mixingposition. Specifically, the flanged end 22 is shown resting snugly inthe well 14 of the base 12. A filter 24, a filter grid 26, and an outletstem 28 are shown, but are not typically in use when themixing/filtering chamber is in the mixing position shown. A port 38 ispresent for transferring whole blood and/or aggregating agent into themixing/filtering chamber 20. A pressure-reducing vent 40 is also presentfor allowing air to escape when displaced by the transfer of whole bloodinto the mixing/filtering chamber 20. Both the vent 40 and the port 38can be equipped with stoppers or valves for preventing the outflow ofwhole blood when the mixing/filtering chamber 20 is in the filteringposition. Though only one port and one vent are shown, it is understoodthat multiple ports and/or vents may be present. For example, separateports can be used for transferring whole blood and aggregating agentinto the mixing/filtering chamber. Alternatively, the whole blood andaggregating agent can be mixed prior to insertion into themixing/filtering chamber 20. Furthermore, the aggregating agent may bepredispensed in the mixing/filtering chamber prior to addition of blood.

[0034] Once the whole blood and aggregating agent are present in themixing/filtering chamber 20 at a desired blood level 42, a magnetic stirbar 32 can be rotated at flanged end 22 using a motor magnet 34controlled by a microprocessor (not shown). A Peltier chip fortemperature control and timer-alarms can also be present within base 12or longitudinal support 16, if desired. In the embodiment shown, themotor magnet 34 is located at the bottom of well 14. Any other stirringor mixing configuration can also be used that is gentle enough to mixthe whole blood with an aggregating agent without substantially damagingor degranulating platelets, but vigorous enough to thoroughly mix theaggregating agent with the whole blood. A stir bar grid 36 is alsopresent to prevent the stir bar 32 from falling onto the filter 24 whenthe chamber 20 is inverted as shown in FIG. 3.

[0035] Referring now to FIG. 3, the mixing/filtering chamber 20 is shownin a filtering position. The mixing/filtering chamber 20 is held in thisposition as the chamber is inserted in aperture (not shown) with flangedend 22 resting on flat surface (not shown). The stir bar 32 is preventedfrom falling into the aggregated whole blood by the stir bar grid 36.

[0036] The filter 24, filter grid 26, stem 28, valve 30, and a syringe44 are now rendered useful with the mixing/filtering chamber 20 in theposition shown in FIG. 3. Specifically, whole blood is drawn through thefilter 24 by creating negative pressure by opening valve 30 andpartially withdrawing the plunger of syringe 44. Though a syringe isshown, any pump device can be used. The filter 24 can include one ormore filters with nominal pore-sizes ranging from 15 to 500 μm. Further,the filter can be designed to capture aggregated platelets whileallowing the passage of non-aggregated cells, e.g. red blood cells andleukocytes, residual aggregating agent, and plasma. The filter can alsoconsist of a removable biodegradable filter, which can be configured tocapture aggregates, and be applied directly (after washing if desired)to the wound site with little or no further processing.

[0037] A filter grid 26 can be present to prevent the filter 24 fromgetting too close to the stem 28, thus maintaining a larger surface areaof filter 24 functional for its intended purpose. The filter 24 haslarge enough pores to allow non-aggregated blood cells through, butsmall enough pores to prevent aggregated platelets from passing. Thus,aggregated platelets can be trapped on the filter, and substantially allof the plasma, leukocytes, and red blood cells can be removed.

[0038] Referring generally to FIGS. 1 to 3, platelet concentratesprepared according to the methods and systems described herein can beprepared by transferring a desired volume of blood to themixing/filtering chamber via an infusion port 38 to attain a desiredblood level 42. Inside air can be vented through an air vent 40. Anaggregating agent can either be present when the blood is transferred tothe chamber, or can be placed with the blood once in the chamber. Theaggregating agent and the whole blood can now be manually,semi-automatically, or automatically mixed. It is to be noted that thechamber in which mixing is accomplished is designed to effectivelyinduce platelet aggregation in whole blood without releasing boundgrowth factors. Thus, mixing should occur that is gentle enough toreduce the release of growth factors, and vigorous enough to promoteadequate aggregation. A stable and relatively fixed, rigid, semi-rigidor moldable partially collapsible chamber can be used to reproduciblycontrol mixing patterns and shear rates for whole blood mixing withaggregation agonist(s) to achieve appropriate levels of plateletaggregation.

[0039] Once adequate mixing has occurred, the platelet aggregates formedin the whole blood are passed through a filter. This is done in thepresent embodiment simply by inverting the mixing/filtering chamber asshown in FIG. 3. Preferably, the mixing/filtering chamber is alsostabilized in the manner previously described. Filtration can occur asgravity forces the blood through the filter 24 and into the stem 28 forremoval. However, other methods can be used to cause flow across thefilter as is desired. This flow can be created manually with a syringe,or by connecting to an evacuated chamber, or automatically with a helpof a pump or linear actuator. Further, though not required,centrifugation can be used to increase the downward force through thefilter and out through the stem. Optionally, a control valve 30 and afilter grid 26 can be used to optimize retention of platelet aggregatesand effective removal of other blood components.

[0040] Filtered blood (devoid of aggregates) can then be collected in aholding receptacle. For example, a syringe 44 can be used for theholding receptacle. This blood can then be returned to the patient,stored (such as for generation of plasma or serum for use as a possiblesubstrate), or disposed.

[0041] Once the non-aggregated portion of the blood has been removed,the filter with platelet aggregates can be rinsed with essentiallyroom-temperature saline (15-25° C.) by manual or automated aspiration.In one embodiment, the saline solution can be injected using a syringethrough the stem 28 that is controlled by the valve 30, or through stem38. Such a rinsing or washing can remove trapped or otherwise presentplasma, other blood cells, and/or residual traces of aggregating agent.The wash can then be collected in another waste container, such asanother syringe, for disposal.

[0042] To collect or concentrate the platelets, the mixing/filteringchamber can then be soaked and aspirated manually or automatically,again through the stem 28 with appropriate fluid, e.g., physiologicalsolution such as saline, plasma, or other isotonic salt solution, todeaggregate the growth factor-containing platelet aggregates.Deaggregated platelets can then be recovered in a collection receptacle,such as yet another syringe or other pump device, and thus, be madeready for use. The harvested platelet concentrate can be mixed with anadjuvant and delivered to the desired site.

[0043] Referring to FIG. 4, an alternative system, indicated generallyat 48, is shown. In this embodiment, a mixing chamber 50 is filled withwhole blood and an aggregating agent through an inlet port 54 to adesired blood level 52. A mixing mechanism 56, which in this case is astirring bar, is present for mixing the whole blood with the aggregatingagent. A conduit 58 is used to transport the aggregated whole blood fromthe mixing chamber 50 to a filtering chamber 62. A valve 60 is presentto prevent flow through the conduit in one or both directions when flowis not desired. Once the aggregated whole blood is in the filteringchamber 62, it is pulled through a porous filter 64 having pore sizesand material properties as previously described, for example. In thepresent embodiment, the filter is in a pleated arrangement, providingincreased surface area if desired. Aggregated platelets larger than apredetermined size will collect on the filter as residual whole bloodcomponents, e.g., plasma, leukocytes, erythrocytes, etc., are allowed topass.

[0044] In the present embodiment, a series of syringes 70, 72, 74 havingdifferent purposes are present and attached to a filter port 76 througha valve 68. The valve 68 can be used to selectively utilize one of thesyringes when desired. If a similar pump system such as provided theseries of syringes 70, 72, 74 are desired for use between the mixingchamber 50 and the filter chamber 62, then a valve port 78 can bepresent as well.

[0045] In one embodiment, a first syringe 70 can be used to draw thewhole blood through the mixing and filtering portions of the system 48.Ultimately, first syringe 70 is used to create the negative pressuredesired for flow of the whole blood through the system 48. The firstsyringe 70 is also used to collect residual blood components notcollected in the filter 64 as previously described. By turning valve 68such that fluid communication between the second syringe 72 and the restof the system 48 can be effected, an aspirating step can occur whereinthe aggregated platelets collected in the filter can be cleaned, such aswith saline or another physiological solution, as will be described morefully hereinafter. Still further, the valve 68 can be oriented forfunctionality of the third syringe 74. The third syringe 74 can be usedto inject deaggregating agent into the filtering chamber 62, as willalso be described hereinafter. Though the pumping, aspirating, anddeaggregating systems shown in this embodiment is a syringe/valvesystem, other systems could also be used with similar success.

[0046] Though both of the device systems of FIGS. 1 to 4 have beendescribed with respect to the separation of platelets from whole blood,other platelet suspensions could also be used. For example, theseparation of platelets from platelet rich plasma and/or platelet packscould also benefit from the systems of the present invention.

[0047] A more fully automated substitute for either of the systems canbe used where the blood filtration, washing, deaggregation, and cellularconcentrate collection steps may be accomplished by automated inverting,valving, and aspiration steps controlled by a microprocessor and linearactuator, for example. A manual alternative for the semi-automateddisposable device can also be implemented for use where the mixing ofaggregating agonist with blood is accomplished by simply shaking orrotating a device, or via a plunger that is aspirated to move and mixthe blood (with or without a stationary mixing grid), or using amotor-operated or spring-loaded stirring or agitation mechanism. Theremainder of the process, i.e., filtration, washing, and retrieval (withor without de-aggregation) can be accomplished manually, orautomatically, as would be known by one skilled in the art after readingthe present disclosure.

[0048] The systems and methods of the present invention allow for theisolation of platelets from whole blood in a device (preferably withoutthe use of a centrifuge) such that growth factors contained within thecells are retained. Underlying elements of the process can involve theisolation of platelets from whole blood by size-selective filtration ofaggregated cells. Platelets are first aggregated by controlled mixing atspecified temperature and time intervals with appropriate aggregatingagents prior to separation by filtration. Platelet aggregates retainedon a filter can then be deaggregated and recovered under conditions thatpreserve their growth factor content. Platelets, when combined with asubstrate, provide a preparation with dispersed cells that can releasegrowth factors on-site immediately and/or over an extended period oftime.

[0049] In another embodiment, a method of stabilizing and healing ananeurysm can comprise the steps of (a) identifying an aneurysm sac; (b)providing a wound healant comprising isolated platelets suspended in thephysiological solution, and a clinical carrier substrate configured forcarrying the platelets; and (c) causing the wound healant to contact theaneurysm sac. The contact can occur by filling the sac with the woundhealant, either fully or partially. Appropriate substrates can includecoils or gels, for example. Additionally, seed cells can also beincorporated into the wound healant, including fibroblasts and smoothmuscle seed cells.

[0050] The following preferred embodiments of another method of thepresent invention are provided, but are not meant to be limiting. Stepsfor obtaining and delivering concentrated platelets can include bloodcollection, platelet aggregation, separation of aggregates from blood,washing platelet aggregates to eliminate excess agonist and other cells,de-aggregation and collection of platelets, and delivery of platelets toa wound site.

[0051] Blood Collection

[0052] Therapeutic wound healing concentration requirements and relatedgel volume needs will dictate preoperative, intraoperative, or bedsideblood collection of about 10 to 200 ml, based on anticipated need. Thepatient's blood can be collected via venapuncture into a container withpre-dispensed anticoagulant, e.g., sodium citrate. The patient's bloodmay be processed immediately or may be stored at room temperature for upto a few hours. The processing of the blood can begin as soon as from 10to 15 minutes before growth factor delivery is desired, the timing beinglimited on the low end by the time it takes to carry out an effectiveseparation and wound healant preparation.

[0053] Use of patient's fresh autologous blood is preferred, and islikely to result in the optimum wound healant for that patient. However,stored blood, e.g., from a blood bank, may be used, although bloodstored for longer than 6 hours may carry the risk of lower platelet,and/or growth factor recovery.

[0054] Platelet Aggregation.

[0055] Once collected, the blood can be transferred to a processingdevice, such as one previously described. A first step in the processinvolves stimulating the platelets to form aggregates under controlledconditions. This can be achieved by the expulsion of prepackagedconcentrations of platelet agonist into blood at a functionaltemperature followed by controlled mixing for a functional amount oftime. Acceptable agonists for platelet aggregation can include thrombin,ristocetin, arachidonic acid, collagen, epinephrine, and ADP. Though allof the above mentioned agonists are functional, their use in somecircumstances may result in the release and loss of granular contents(such as growth factors), or have other adverse processing effects uponaggregation. This aside, certain positive characteristics may outweighperceived negative affects. For example, collagen may be desired inspecific circumstances where growth factors are preferably to bedelivered in a collagen substrate. In one embodiment, the use of from 10μM to 100 μM of ADP can be preferred for platelet aggregation. Bothcollagen and ADP have some inherent growth promoting features. Inanother embodiment, the use of collagen or epinephrine can be preferredfor platelet aggregation.

[0056] Though any functional temperature and mixing time can be used,preferred temperatures for aggregation can be from 15° C. to 42° C., anda more preferred temperature range can be from 20° C. to 37° C.Preferred mixing times can be from 15 to 300 seconds, and more preferredmixing times can be from 60 to 180 seconds. If a stir bar is used formixing, any functional RPM rate can be used, though from 60 to 3000 RPMsprovides a range, and from 200 to 1000 1500 RPMs provides a preferredrange for stirring. This speed may vary depending on the geometry of themixing chamber and geometry of the stir bar. The temperature, time, andstirring force for mixing can be optimized with respect to a specificsystem, as would be ascertainable by one skilled in the art afterreading the present disclosure. In one embodiment, these parameters canbe optimized to create platelet aggregates with nominal dimensions ofover approximately 15 μm for eventual filtration.

[0057] The platelet aggregation process should not be allowed to proceedbeyond specific time points (typically, 3 minutes or less) dependentupon the aggregating agent utilized. Controlling the elapsed time foraggregation can minimize the risk of growth factor leakage. For example,as disclosed in Mohammad S F, Am J Pathol. 79: 81-94. 1975, a loweraggregation time may be preferred by restricting cell aggregation to thefirst phase of aggregation (for ADP aggregation), and interrupting theprocess before the second phase of aggregation (the stage during whichplatelet granular contents are released). Conducting the aggregationprocess at room temperature, or at temperatures less than 37° C., mayalso provide some protection against over-aggressive aggregation ofplatelets that could lead to release of granular contents (such asgrowth factors) that may occur at higher temperatures, e.g., 37° C.However, this could potentially reduce the efficiency of the aggregatingprocess leading to slightly lower yields of platelets compared withaggregation at 37° C. Mixing dynamics of agonist and blood should alsobe considered for controlled aggregation. If mixing is too gentle, allsingle platelets may not aggregate. If mixing is too aggressive, highshears and violent collisions may disrupt formed aggregates, thusdamaging the cells and releasing the cytoplasmic contents includinggrowth factors. Optimal ranges of time of aggregation (˜1 to 3 minutes),temperature (22° C. to 37° C.), and mixing, e.g. rotational speeds inthe order of 200 to 1500 RPM for proper mixing accomplished by astir-bar in a cylindrical cup-like chamber, that results in maximumaggregation of platelets and minimum loss of growth factors are desired.

[0058] Separation of Aggregates from Blood

[0059] After agonist mixing is stopped, blood with cellular aggregatesis immediately passed through a filter assembly e.g. filter(s) of singleor multiple stages with nominal pore size(s) between 15 and 500 μm.Though pore sizes from 100 to 500 μm are functional, preferably, thepore size can be from 15 to 100 μm. This being said, any pore size thatallows for the retention of cell aggregates and passage of single bloodcells and plasma is considered functional.

[0060] Washing Platelet Aggregates to Eliminate Excess Agonist and Cells

[0061] Aggregates retained on the filter may be rinsed with wash fluid,such as room-temperature saline, to remove residual agonist, plasmaproteins, and loosely trapped cells, leaving behind the aggregatedplatelets. The washing fluid can be of any functional temperature and ofany functional pH. However, the temperature is preferably from 15° C. to30° C., and more preferably from 18° C. to 25° C. The pH level of thewashing fluid can be from 5.5 to 8, but is preferably from 6 to 8.

[0062] Deaggregation and Collection of Platelets

[0063] In a preferred process, deaggregation can be carried out forplatelet aggregates captured in the filter assembly. This allows forsubsequent dispersion of cells or small aggregates in a desired mediumwhen the cells are delivered to a wound site. Deaggregation, and theconditions under which it is carried out, may also lead to effectiveretention of growth factors inside the cells, as the aggregation processis reversed before degranulation ensues. Deaggregation may also resultin higher cell yields because retrieval of deaggregated cells may beless demanding and more efficient than removing filter-bound intactaggregates.

[0064] To reverse aggregation, aggregates captured on the filter can besoaked in a specified volume of a medium of a desired functionaltemperature and pH. Such appropriate deaggregating mediums can includesaline, ACD-saline, or other salt solutions with additives to facilitatedeaggregation, e.g., apyrase, aspirin, IIb/IIIa antagonists, otherplatelet function suppressors, plasma, albumin, or the like. This can beaccompanied by frequent mixing, e.g., aspiration with a syringe, and/oragitation, e.g., vibration, for a functional time period to facilitatedeaggregation and producing single cells or small aggregates. Preferredtemperature for this process can be from 18° C. to 42° C., with morepreferred temperatures being from 33° C. to 37° C. Preferred pH levelscan be from 5.5 to 8, with a more preferred pH range from 6 to 8. Anyfunctional time for deaggregation can be implemented, though from 30 to1000 seconds is considered to be preferred, with from 30 to 300 secondsbeing more preferred.

[0065] In an alternative embodiment, by aggressive aspiration, cellaggregates can be separated and recovered without deaggregation withinthe sealed filter compartment, followed by retrieval of both bound andexpelled growth factors.

[0066] In another embodiment, aggregates may be left on the filter,platelet growth factors expelled from the aggregates by any means known,e.g., degranulation, and collected in a desired volume for delivery tothe wound site.

[0067] Delivery of Platelet Concentrates to the Wound Site

[0068] The suspensions obtained from this process may be combined with awide variety of clinical wound treatment adjuvant substrates fordelivery to the wound site. Specific substrate properties can beselected to tailor growth factor delivery to promote healing in soft,hard, and/or connective tissue. The concentrated growth factor-ladencells or solutions from this process can readily be combinedintraoperatively with a variety of dressing and delivery substratesincluding: hydrogels, hyaluronic acid, gelatins, fibrin and fibrinogen,collagen, alginate, albumin, chitosan and other sponges, surgical foams,plasma or serum along with antimicrobials and pharmaceuticals, etc.

EXAMPLES

[0069] The following example illustrates a preferred embodiment of theinvention that is presently known. However, other embodiments can bepracticed that are also within the scope of the present invention.

Example 1 Separation of Platelets from Whole Blood

[0070] A separation of platelets from whole blood was carried out by thefollowing process: About 10 ml of whole blood was collected from 4 humansubjects by venapuncture into a syringe having a predispensedanticoagulant contained therein. To the collected whole blood was added100 μM of ADP as an aggregating agent. The whole blood and aggregatecombination was mixed in a chamber with a stir bar for 90 sec at 37° C.Once mixing was stopped, the blood with cellular aggregates was filteredthrough a filter assembly having pore sizes ranging from 20 μm to 100 μmunder negative pressure exerted by a syringe. The filtered aggregateswere washed with 30 ml of 18° C. saline for 1 minute. Next, the washedaggregates were incubated with a saline-ACD solution at 37° C. withgentle aspiration for 3-5 minutes. The saline-ACD solution havingsubstantially deaggregated growth-factor containing platelets were thencollected as a suspension.

[0071] Specifically, to assess the potential effectiveness of the aboveprocess of isolation of platelets, the following experiments wereconducted: the yield of platelets harvested from human blood wasdetermined and quantified (Example 2); aspects of the functionalintegrity of the platelets was determined (Example 3); and the presenceof one of the most recognized growth factors, PDGF-AB, as arepresentative growth factor was determined (Example 4).

Example 2 Determination of Platelet Yield

[0072] A platelet recovery assay was performed by placing a dilution ofa platelet suspension, prepared in accordance with Example 1, in ahemocytometer where the number of platelets were counted using a phasecontrast microscope or with the help of an electronic particle counter.Platelets recovered were compared with platelets recovered using aconventional centrifugation method of the prior art. Hemocytometercounts showed near complete recovery of platelets using the aggregation,filtration, and deaggregation method of the present invention. Theresults were quantified and are shown in FIG. 5. The waste filtrate fromthis process contained very few platelets in all cases indicating thataggregation and filtration process was very efficient in harvestingplatelets from whole blood. It is worth noting that two of the foursubjects that were part of this study were taking aspirin and/or calciumchannel blockers (as anti-hypertension medication). Aspirin is a knownsuppressor of platelet aggregation, but platelets aggregated well usingthe methods described in the present invention and good recovery wasobserved. This being said, some patients with severe plateletdeficiencies, or thrombocytopenia, or patients using potent plateletantagonists may not be preferred candidates for this process becausetheir platelet counts may be too low or their functional integrity maybe compromised. Such candidates may benefit more from plateletscollected from a blood donor.

Example 3 Determination off Unctional Integrity

[0073] To assess functional integrity, platelets recovered in accordancewith Example 1 were added to autologous platelet poor plasma, incubatedfor 15 minutes at 37° C., and the function of platelets assessed in aBIO/DATA turbidometric platelet aggregometer using 50 μM of ADP as theaggregating agent. Platelets recovered by the present invention werecompared with platelets in platelet rich plasma obtained by conventionalcentrifugation. The comparison of platelet function in a turbidometricaggregometer showed virtually identical platelet aggregation profilesbetween the platelets recovered by centrifugation, and those recoveredby the present invention. FIG. 6 depicts these results. This suggeststhat the functional integrity of harvested and concentrated plateletsobtained by the process of the present invention was not compromisedwhen compared to a prior art method.

Example 4 Determination of Presence of PDGF-AB

[0074] To determine platelet-derived growth factor (PDGF-AB) presence inplatelets concentrated as in Example 1, a chromogenic ELISA method(Quantikine, R&D systems) was utilized. Concentrated platelets obtainedby the centrifugation method served as the reference (control) for validcomparisons. Functional viability of the growth factors contained inrecovered platelets was assessed by measuring enhancement of humanaortic smooth muscle cell proliferation. ELISA results indicatedpreservation of PDGF-AB in platelets recovered by this process similarto that of PDFG-AB from platelets recovered by centrifugation, as isshown in FIG. 7. This suggests that process steps outlined herein ensurethat internal contents of the dense granules in the platelets, e.g.,growth factors, are not substantially expelled. The negative control(platelet poor plasma) expressed virtually no PDGF-AB, which suggestsvalid experimental conditions. The full recovery of PDGF-AB in theplatelets harvested by a process of the present invention indicates thatother growth factors (PDGF-AA, TGF, VEGF, FGF, etc.) contained inplatelets may likewise be preserved during the recovery process.

Example 5 Delivery of Platelet Concentrates to Smooth Muscle Cells

[0075] The platelet suspension derived by the method of Example 1enhanced human aortic smooth muscle cell proliferation by 24% comparedwith the blank buffer control. The results were obtained from twosubjects with samples analyzed in triplicate with a MTT assay.

[0076] It is to be understood that the above-referenced arrangements areonly illustrative of the application for the principles of the presentinvention. Numerous modifications and alternative arrangements can bedevised without departing from the spirit and scope of the presentinvention while the present invention has been shown in the drawings andfully described above with particularity and detail in connection withwhat is presently deemed to be the most practical and preferredembodiments(s) of the invention, it will be apparent to those ofordinary skill in the art that numerous modifications can be madewithout departing from the principles and concepts of the invention asset forth in the claims.

What is claimed is:
 1. A method for separating platelets from wholeblood, comprising: providing whole blood including platelets,leukocytes, erythrocytes, and blood plasma; selectively aggregating theplatelets using an aggregating agent to form platelet aggregates thatare larger in size than said leukocytes and said erythrocytes; andsubstantially separating the platelet aggregates from the leukocytes,erythrocytes, and blood plasma.
 2. A method as in claim 1, wherein theplatelet aggregates are washed with an isotonic solution after theseparating step.
 3. A method as in claim 1, wherein the plateletaggregates are at least partially deaggregated after the separatingstep.
 4. A method as in claim 1, wherein the platelet aggregates aresuspended in a physiological isotonic solution after the separatingstep.
 5. A method as in claim 3, wherein the deaggregated platelets areconcentrated to a therapeutic level for delivery to a wound site.
 6. Amethod as in claim 1, wherein the separating step is carried out withoutthe use of centrifugation.
 7. A method as in claim 1, wherein theseparating step is by a filtering step.
 8. A method as in claim 1,wherein the separating step is by a sedimentation step.
 9. A method asin claim 1, wherein the aggregating agent is selected from the groupconsisting of thrombin, ristocetin, arachidonic acid, collagen,epinephrine, adenosine di-phosphate, and combinations thereof.
 10. Amethod as in claim 9, wherein the aggregating agent is adenosinediphosphate.
 11. A method as in claim 2, wherein the platelets arewashed with a physiological solution at a temperature from about 18° C.to 25° C. and for about 1 to 3 minutes to remove residual componentsincluding agonists, red blood cells, white blood cells, and plasmaproteins.
 12. A method as in claim 1, further comprising the steps ofaspirating the platelet aggregates after separation with physiologicalsolution to deaggregate and resuspend the platelets while minimizingdegranulation, thereby recovering single cells or small cell aggregates,said physiological solution being configured to preserve growth factorsof the platelets.
 13. A method as in claim 12, wherein the aspiratingstep is carried out with plasma.
 14. A method as in claim 12, whereinthe aspirating step is carried out using a member selected from thegroup consisting of ACD-saline and albumin solution.
 15. A method as inclaim 12, wherein the aspirating step occurs under controlledtemperatures from 33° C. to 37° C., and at a pH from about 6 to
 8. 16. Amethod as in claim 7, wherein the step of filtering is conducted with abiodegradable filter that can be placed directly on a wound site.
 17. Amethod as in claim 16, wherein the biodegradable filter comprises amaterial selected from the group consisting of polyglycolic acid,polylactic acid, polypeptide, collagen, and combinations thereof.
 18. Amethod as in claim 1, wherein the separation occurs within 15 minutesfor planned or emergency reinfusion or transfusion of the platelet. 19.A method as in claim 1, wherein the step of selectively aggregating theplatelets is enhanced by mixing the whole blood and the aggregatingagent at a temperature from 20° C. to 37° C. for about 60 to 180seconds.
 20. A method for separating platelets from a plateletsuspension, comprising: selectively aggregating platelets in a plateletsuspension using an aggregating agent to form platelet aggregates; andseparating the platelet aggregates from platelet suspension byfiltration.
 21. A method as in claim 20, wherein the separating stepoccurs without centrifugation.
 22. A method as in claim 20, wherein theaggregating agent is selected from the group consisting of thrombin,ristocetin, arachidonic acid, collagen, epinephrine, adenosinedi-phosphate, and combinations thereof.
 23. A method as in claim 20,wherein the platelet aggregates are washed with a physiological solutionafter filtration.
 24. A method as in claim 20, wherein the plateletaggregates are at least partially deaggregated after filtration.
 25. Amethod as in claim 20, wherein the platelet aggregates are suspended ina physiologically neutral solution after filtration.
 26. A method as inclaim 20, wherein the platelet suspension is platelet rich plasma.
 27. Amethod as in claim 20, wherein the platelet suspension is whole blood.28. A method as in claim 20, wherein the platelet suspension is aplatelet pack.
 29. A device for separating platelets from whole blood,comprising: a mixing/filtering chamber configured for mixing whole bloodand an aggregating agent to form platelet aggregates when positioned ina first orientation, and further configured for collecting the plateletaggregates when positioned in a second orientation; at least one inletport for transferring whole blood and the aggregating agent into themixing/filtering chamber; a mixing mechanism for mixing the whole bloodand the aggregating agent when the mixing/filtering chamber ispositioned in the first orientation; a filter for collecting theplatelet aggregates when the mixing/filtering chamber is positioned inthe second orientation; and an outlet port for removing components ofthe whole blood that are not collected in the filter.
 30. A device as inclaim 29, wherein the mixing mechanism is configured to optimize mixingto prevent substantial premature release of growth factor contents fromthe platelets.
 31. A device as in claim 29, wherein the filter has apore size from 15 to 500 uM.
 32. A device as in claim 31, wherein thepore size is from 15 to 100 um.
 33. A device as in claim 29, wherein theoutlet port is also used for injecting a physiological solution into themixing/filtering chamber for washing.
 34. A device as in claim 29,wherein the mixing mechanism is an electromagnetic motor and a magneticstir bar.
 35. A device as in claim 29, wherein the filter comprises amaterial selected from the group consisting of metal, polymer,biomaterial, biodegradable material, and combinations thereof.
 36. Adevice as in claim 35, wherein the filter comprises a material selectedfrom the group consisting of stainless steel, nylon,poly-tetra-fluoro-ethylene, polyester, hyaluronic acid, and combinationsthereof.
 37. A device for separating platelets from whole blood,comprising: a mixing chamber configured for receiving and mixing wholeblood and an aggregating agent to form platelet aggregates and residualblood components; a filtering chamber configured for collecting plateletaggregates and allowing the residual blood components to substantiallypass, said filtering chamber comprising a porous filter; a valvedisposed between the mixing chamber and the filtering chamber forholding the whole blood and the aggregating agent in the mixing chamberduring mixing, and for allowing flow of the platelet aggregates and theresidual blood components from the mixing chamber to the filteringchamber for filtering; and an outlet port configured for removingcomponents of the whole blood that are not collected in the filter. 38.A device as in claim 37, wherein the mixing chamber includes a mixingmechanism optimized to prevent substantial premature release of growthfactor contents from the platelets.
 39. A device as in claim 37, whereinthe porous filter has a pore size from 15 to 500 um.
 40. A device as inclaim 39, wherein the pore size is from 15 to 100 um.
 41. A device as inclaim 37, wherein the outlet port is also used for injecting aphysiological medium into the filtering chamber for washing theaggregates.
 42. A device as in claim 37, wherein the outlet port is alsoused for injecting a deaggregating agent into the filtering chamber fordeaggregating the aggregates.
 43. A device as in claim 37, wherein themixing device is an electromagnetic motor and a magnetic stir bar.
 44. Adevice as in claim 37, wherein the filter comprises a material selectedfrom the group consisting of metal, polymer, biomaterial, biodegradablematerial, and combinations thereof.
 45. A device as in claim 44, whereinthe filter comprises a material selected from the group consisting ofstainless steel, nylon, poly-tetra-fluoro-ethylene, polyester,hyaluronic acid, and combinations thereof.
 46. A device as in claim 37,wherein the removal of components not collected on the filter isprovided by a syringe acting on the outlet port.
 47. A device as inclaim 41, wherein the physiological medium is injected into thefiltering chamber with a syringe.
 48. A device as in claim 42, whereinthe deaggregating agent is injected into the filtering chamber with asyringe.
 49. A device as in claim 37, wherein the device is fullyautomated.
 50. A wound healant for direct application to tissue,comprising: a physiological solution free of degranulating agent;isolated platelets suspended in the physiological solution; and aclinical carrier substrate configured for carrying the platelets to awound site.
 51. A wound healant as in claim 50, wherein the clinicalcarrier substrate is selected from the group consisting of biomaterials,alginate, collagen, gelatin, hydrogel, hyaluronic acid, bone fillers,calcium phosphate, chitosan, natural sponges, surgical foams, fibrinogensolution, plasma, fibrin glue, and combinations thereof.
 52. A woundhealant as in claim 50, wherein the platelets with contained growthfactors are present at a concentration greater than three times normallevels compared to that present in whole blood.
 53. A wound healant asin claim 50, wherein the physiological solution is further free of serumor plasma proteins.
 54. A wound healant as in claim 50, wherein theplatelets are present as single cells or small aggregates of cells andare distributed throughout the clinical carrier substrate, and notfurther treated with a degranulating agent, thereby providing a uniformdispersed and prolonged release of growth factors.
 55. A wound healantas in claim 50, prepared for delivery using a polyglycolic acid orpolyester patch.
 56. A wound healant as in claim 50, prepared fordelivery using a gel seal suture.
 57. A wound healant as in claim 50,further comprising a hemostatic sealant to contribute growth-promotingproperties of the wound healant.
 58. A wound healant for application totissue, comprising a combination of platelet aggregates and singleplatelets, said platelet aggregates and said single platelets beingsuspended in a physiological solution and carried by clinical carriersubstrate.
 59. A wound healant as in claim 58, wherein the clinicalcarrier substrate is selected from the group consisting of biomaterials,alginate, collagen, hydrogel, hyaluronic acid, calcium phosphate,fibrinogen solution, clotted plasma, fibrin glue, and combinationsthereof.
 60. A wound healant as in claim 58, wherein the physiologicalsolution is substantially free of serum or plasma proteins.
 61. A woundhealant as in claim 58, wherein a mixture of single platelet cells,aggregates, and expelled platelet growth factors are dispersedthroughout the clinical carrier substrate, thereby providing animmediate growth factor release and prolonged growth factor release. 62.A wound healant as in claim 58, prepared for delivery using apolyglycolic acid patch or a polyester patch.
 63. A wound healant as inclaim 58, prepared for delivery using a gel seal suture.
 64. A woundhealant as in claim 58, further comprising a hemostatic sealant tocontribute growth-promoting properties of the wound healant.
 65. A woundhealant as in claim 58, wherein the majority of the platelets are partof the platelet aggregates.
 66. A wound healant as in claim 58, whereinthe majority of the platelets are the single platelets.
 67. A method ofstabilizing and healing an aneurysm, comprising: identifying an aneurysmsac; providing a wound healant comprising isolated platelets suspendedin the physiological solution, and a clinical carrier substrateconfigured for carrying the platelets; and causing the wound healant tocontact the aneurysm sac.
 68. A method as in claim 67, wherein the stepof causing the wound healant to contact the aneurysm sac is by fillingthe aneurysm sac with the wound healant.
 69. A method as in claim 68,wherein the aneurysm sac is partially filled.
 70. A method as in claim67, wherein the wound healant further comprises seed cells.